Ethanol production

ABSTRACT

Production of ethanol in a thermophilic bacterium can be achieved by transformation of the bacterium with a heterologous gene encoding pyruvate decarboxylase. The bacterium comprises native alcohol dehydrogenase function and does not comprise a gene encoding a functional lactate dehydrogenase. The bacterium may be grown at elevated temperatures to allow the ethanol to be conveniently removed in a vaporized form from the fermentation medium. Traditional mesophilic microorganisms are incapable of growth at the envisioned elevated temperatures.

This invention relates to the production of ethanol as a product ofbacterial fermentation. In particular, the invention relates to ethanolproduction by thermophilic strains of Bacillus sp.

Many bacteria have the natural ability to metabolise simple sugars intoa mixture of acidic and neutral fermentation products via the process ofglycolysis. Glycolysis is the series of enzymatic steps whereby the sixcarbon glucose molecule is broken down, via multiple intermediates, intotwo molecules of the three carbon compound pyruvate. The glycolyticpathways of many bacteria produce pyruvate as a common intermediate.Subsequent metabolism of pyruvate results in a net production of NADHand ATP as well as waste products commonly known as fermentationproducts. Under aerobic conditions, approximately 95% of the pyruvateproduced from glycolysis is consumed in a number of short metabolicpathways which act to regenerate NAD⁺ via oxidative metabolism, whereNADH is typically oxidised by donating hydrogen equivalents via a seriesof steps to oxygen, thereby forming water, an obligate requirement forcontinued glycolysis and ATP production.

Under anaerobic conditions, most ATP is generated via glycolysis.Additional ATP can also be regenerated during the production of organicacids such as acetate, NAD⁺ is regenerated from NADH during thereduction of organic substrates such as pyruvale or acetyl CoA.Therefore, the fermentation products of glycolysis and pyruvatemetabolism include organic acids, such as lactate, formate and acetateas well as neutral products such as ethanol.

The majority of facultatively anaerobic bacteria do not produce highyields of ethanol either under aerobic or anaerobic conditions. Mostfacultative anaerobes metabolise pyruvate aerobically via pyruvatedehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Underanaerobic conditions, the main energy pathway for the metabolism ofpyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate andacetyl-CoA. Acetyl-CoA is then converted to acetate, viaphosphotransacetylase (PTA) and acetate kinase (AK) with theco-production of ATP, Or reduced to ethanol via acetaldehydedehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order tomaintain a balance of reducing equivalents, excess NADH produced fromglycolysis is re-oxidised to NAD⁺ by lactate dehydrogenase (LDH) duringthe reduction of pyruvate to lactate. NADH can also be re-oxidised byAcDH and ADH during the reduction of acetyl-CoA to ethanol but this is aminor reaction in cells with a functional LDH. Theoretical yields ofethanol are therefore not achieved since most acetyl CoA is converted toacetate to regenerate ATP and excess NADH produced during glycolysis isoxidised by LDH.

Ethanologenic organisms, such as Zymomonas mobilis and yeast, arecapable of a second type of anaerobic fermentation commonly referred toas an alcoholic fermentation in which pyruvate is metabolised toacetaldehyde and CO₂ by pyruvate decarboxylase (PDC). Acetaldehyde isthen reduced to ethanol by ADH regenerating NAD⁺ Alcoholic fermentationresults in the metabolism of 1 molecule of glucose to two molecules ofethanol and two molecules of CO₂. The genes which encodes both of theseenzymes in Z. mobilis have been isolated, cloned and expressedrecombinantly in hosts capable of producing high yields of ethanol viathe synthetic route described above. For example; U.S. Pat. No.5,000,000 and Ingram et al (1997) Biotechnology and Bioengineering 58,Nos. 2 and 3 have shown that the genes encoding both PDC (pdc) and ADH(adh) from Z. mobilis can be incorporated into a “pet” operon which canbe used to trans form Escherichia coli strains resulting in theproduction of recombinant E. coli capable of co-expressing the Z.mobilis pdc and adh. This results in the production of a syntheticpathway re-directing E. coli central metabolism from pyruvate to ethanolduring growth under both aerobic and anaerobic conditions. Similarly,U.S. Pat. No. 5,554,520 discloses that pdc and adh from Z. mobilis canboth be integrated via the use of a pet operon to produce Gram negativerecombinant hosts, including Erwina, Klebsiella and Xanthomonas species,each of which expresses the heterologous genes of Z. mobilis resultingin high yield production of ethanol via a synthetic pathway frompyruvate to ethanol.

U.S. Pat. No. 5,482,846 discloses the simultaneous transformation ofGram positive Bacillus sp with heterologous genes which encode both thePDC and ADH enzymes so that the transformed bacteria produce ethanol asa primary fermentation product. There is no suggestion that the bacteriamay be transformed with the pdc gene alone.

A key improvement in the production of ethanol using biocatalysts can beachieved if operating temperatures are increased to levels at which theethanol is conveniently removed in a vapourised form from thefermentation medium. However, at the temperatures envisioned,traditional mesophilic microorganisms, such as yeasts and Z. mobilis,are incapable of growth. This has led researchers to consider the use ofthermophilic, ethanologenic bacteria as a functional alternative totraditional mesophilic organisms. See EP-A-0370023.

The use of thermophilic bacteria for ethanol production offers manyadvantages over traditional processes based upon mesophilic ethanolproducers. Such advantages include the ability to ferment a wide rangeof substrates, utilising both cellobiose and pentose sugars found withinthe dilute acid hydrolysate of lignocellulose, as well as the reductionof ethanol inhibition by continuous removal of ethanol from the reactionmedium using either a mild vacuum or gas sparging. In this way, themajority of the ethanol produced may be automatically removed in thevapour phase at temperatures above 50° C. allowing the production phaseto be fed with high sugar concentrations without exceeding the ethanoltolerance of the organism, thereby making the reaction more efficient.The use of thermophilic organisms also provides significant economicsavings over traditional process methods based upon lower ethanolseparation costs.

The use of facultative anaerobes also provides a number of advantages inallowing a mixed aerobic and anaerobic process. This facilitates the useof by-products of the anaerobic phase to generate further catalyticbiomass in the aerobic phase which can then be returned to the anaerobicproduction phase.

The inventors have produced sporulation deficient variants of athermophilic, facultatively anaerobic, Gram-positive bacterium whichexhibit improved ethanol production-related characteristics.

This approach has a number of important advantages over conventionalprocesses using both traditional and recombinant mesophilic bacteria,including simplification of the transformation process by using only thepdc gene of Z. mobilis in strains that already produce ethanol and havea ‘native’ adh gene. Expression of pdc has resulted in a significantincrease in ethanol production by the recombinant organism and hasunexpectedly improved the organism's growth characteristics. Recombinantmicroorganisms, which prior to transformation with the pdc gene werehighly unstable and difficult to culture, show significant increases ingrowth and survival rates both aerobically and anaerobically as well asan increase in the rate of ethanol production near to theoreticalyields.

Accordingly, a first aspect of the invention relates to a Gram-positivebacterium which has been transformed with a heterologous pdc gene, butwhich has solely a native alcohol dehydrogenase function. The gene mayencode a functional equivalent of pyruvate decarboxylase Functionalequivalents of pyruvate decarboxylase include expression products ofinsertion and deletion mutants of natural pdc gene sequences.

It is possible that organisms which carry out glycolysis or a variantthereof can be engineered, in accordance with the present invention, toconvert as much as 67% of the carbon in a sugar molecule via glycolysisand a synthetic metabolic pathway comprising enzymes which are encodedby heterologous pdc and native adh genes. The result is an engineeredorganism which produces ethanol as its primary fermentation product.

The Gram-positive bacterium is preferably a Bacillus. The bacterium maybe a thermophile. Where the Gram-positive bacterium is a Bacillus it ispreferably selected from B. stearothermophilus; B. calvodex; B.caldotenax; B. thermoglucosidasius, B. coagulans, B. licheniformis, B.thermodenitrificans, and B. caldolyticus.

A gene encoding lactate dehydrogenase may be inactivated in theGram-positive bacterium of the invention. For example, the lactatedehydrogenase gene may be inactivated by homologous recombination.

The heterologous pdc gene may be from Zymomonas sp, preferably Z.mobilis or may be from yeast e.g. the S. cerevisae pdc 5 gene.

The heterologous gene may be incorporated into the chromosome of thebacterium. Alternatively, the bacterium may be transformed with aplasmid comprising the heterologous gene. Preferably the bacterium istransformed using plasmid is pFC1, more preferably with pFC1-PDC1. Theinvention includes Gram-positive bacteria, preferably a Bacillus spincluding the operon of the invention. The Bacillus sp may be selectedfrom B. stearothermophilus; B. calvodex; B. caldotenax, B.thermoglucosidasius, B. coagulans, B. licheniformis, B.thermodenitrificans, and B. caldolyticus. The operon may be incorporatedinto the genome of the Bacillus. Multiple copies of the PDC operon maybe incorporated into the genome.

In a preferred embodiment of the present invention the Gram-positivebacteria has the heterologous gene operatively linked to the lactatedehydrogenase promoter from Bacillus strain LN (NCIMB accession number41038) so that the heterologous gene is under the control of thepromoter. The sequence of the promoter region from strain LN is shown inFIG. 8.

The invention also provides strains LN (NCIMB accession number 41038);LN-T (E31, E32); TN (NCIMB accession number 41039); TN-P1; TN-P3; LA-S(J8) (NCIMB accession number 41040); LN-D (NCIMB accession number41041); —N-D11 and LN-P1.

According to another aspect of the invention, there is provided arecombinant, sporulation deficient, thermophilic Bacillus which grows atgreater than 50° C. The Bacillus is preferably not B. licheniformis.

A second aspect of the present invention relates to a method ofproducing ethanol using bacteria of the invention maintained undersuitable conditions.

The method may be operated at a temperature between 40-75° C.;preferably at a temperature of 52-65° C.; most preferably at atemperature of 60-65° C.

The present invention also relates to a method of producing L-lacticacid using strain LN.

The present invention also provides a nucleic acid molecule comprisingthe lactate dehydrogenase promoter of strain LN (NCIMB accession number41038). The sequence of a nucleic acid molecule comprising the lactatedehydrogenase promoter of strain LN is shown in FIG. 7. Preferably thenucleic acid molecule comprises a functional fragment of the nucleicacid sequence shown in FIG. 7. A functional fragment is defined as afragment that function as a promoter and enables the expression of anoperably linked gene.

The present invention also provides plasmid pFC1. The structure of thisplasmid is shown schematically in FIG. 8

The present invention also provides plasmid pFC1-PDC1. The structure ofthis plasmid is shown schematically in FIG. 9.

The production of recombinant bacteria in accordance with the inventionwill now be described, by way of example only, with reference to theaccompanying drawings, FIGS. 1 to 10 in which:

FIG. 1 is a schematic representation showing the production of bacterialstrains of the invention;

FIG. 2 illustrates the metabolic pathway whereby sugars are metabolisedto produce ethanol by bacterial strains of the invention;

FIG. 3 is a schematic representation illustrating the method of LDH geneinactivation by single crossover recombination;

FIG. 4 is a schematic representation illustrating the method of LDH geneinactivation by double crossover recombination;

FIG. 5 is a schematic representation illustrating the method of LDH geneinactivation and heterologous gene expression by double crossoverrecombination;

FIG. 6 provides details about the PDC gene and promoter construct;

FIG. 7 provides sequence data about the LDH promoter from Bacillus LN;

FIG. 8 is a schematic disown of the replicative plasmid pFC1;

FIG. 9 is a schematic diagram of the PDC construct 2 coned into the pFC1plasmid; and

FIG. 10 shows the construction of an artificial PDC operon.

The inventors initiated a strain development program to overcomeinherent strain limitations in respect of ethanol production, such asinstability and sporulation under adverse conditions. Physiologicalmanipulation and selection for strains with superior growthcharacteristics has been achieved in continuous culture, whereas a moretargeted genetic approach has been used to engineer strains with greaterstability and superior production characteristics.

In accordance with FIG. 1, all strains were ultimately derived from apreviously isolated Bacillus isolate PSII, a novel, thermophilic,Gram-positive, spore-forming, facultative anaerobe. PSII ferments a widerange of organic compounds from sugars, including hemicelluloses, toorganic acids such as acetate, formate and lactate and small amounts ofethanol at temperatures between 50° C. and 70° C.

Bacillus strains LLD-15, LLD-R, LLD-16 and T13 have been described inEP-A-0370023 and by Amartey et al, (1999) Process Biochemistry 34 No. 3pp. 289-294. Strain LLD-15 (NCIMB12428) arose during attempts to obtainmutants of Bacillus stearothermophilus strain NCA 1503 lacking L-LDHactivity by selecting for suicide substrate resistance (Payton andHartley (1984) Trends in Biotechnology, 2 No. 6). Strain LLD-15 wasassumed to be a variant of B. stearothermophilus NCA1503, but is, infact, derived from PS11. Strain LLD-R arises spontaneously andreproducibly from strain LLD-15 and is selected on plates or duringcontinuous cultures under which it grows more rapidly (i.e. at low pH inmedia containing sugars, acetate and formate). LLD-R produces L-lactateanaerobically and contains high levels of L-LDH, so is therefore,clearly a wild type revertant of the non-lactate-producing LLD-15lesion.

Bacillus strain T13 is an L-lactate deficient mutant of strain LLD-R.Isolation and characterisation of this mutant strain has been describedpreviously by, Javed. M. PhD Thesis, Imperial College, London. Thus,Bacillus strains LLD-15, LLD-16 and T13 are mutants of the originalisolate, PSII in which the ldh gene has been inactivated either viaspontaneous mutation or by chemical mutagenesis and the majorfermentation product, unlike PSII, is ethanol. The lactate dehydrogenasegene mutation in LLD-15, LLD-16 and T13 results from the insertion of atransposon into the coding region of the lactate dehydrogenase generesulting in ldhT geno-type. All three strains are inherently unstablein high sugar concentrations (>2%) and revert back to lactate producingstrains. Strains T13 and LLD-15 revert to T13-R and LLD-R, respectively.Strains LLD-15, LLD-R, LLD-16 and T13 also tend to sporulate underadverse growth conditions such as changes in pH, temperature and mediumcompositions, and during periods of nutrient starvation. Sincesporulation often leads to culture washout in a continuous system, thesestrains are not suitable for large scale industrial use in which processparameters fluctuate.

EXAMPLE 1 Production of LN

Bacillus strain LN was produced from LLD-R as a spontaneous sporulationmutant which arose during nitrogen adaptation in continuous culture.Strain LN is more robust than the parental strains LLD-15 and LLD-Runder vigorous industrial conditions such as low pH, high sugarconcentrations and in crude hydrolysate feed stocks and is more amenableto plasmid transformation. Strain LN is sporulation deficient (spo⁻) andis particularly suitable for the production of high pity L-lactic acid,producing up to 0.4 g of L-lactate/g of glucose at 65° C. This strain issensitive to kanamycin concentrations in excess of 5 μg/ml and an idealhost for genetic manipulation (ldh inactivation and heterologous geneexpression). Strain LN has been deposited under the terms of theBudapest Treaty under accession No. NCIMB 41038.

EXAMPLE 2 Production of Strain LN-T (E31/E32)

Strains E31 and E32 are spontaneous transposon mutants from strain LN.Both strains are lactate deficient and produce up to 0.5 g of ethanol/gof glucose at 65° C. They are non-sporulating and as such more amenableto genetic manipulation than T13.

EXAMPLE 3 Production of Strain LN-S (J8)

Strain LN-S (J8) was produced by a single crossover homologousrecombination event between pUBUC-LDH a temperature sensitive,non-replicative plasmid harbouring an internal region of the ldh geneand the ldh gene on the chromosome (see FIG. 3). This resulted ininactivation of the ldh gene and a lactate negative phenotype. Thisstrain is also sporulation deficient and resistant to kanamycin. It isstable in relatively high sugar concentrations in continuous culture(with 10 g/L of residual sugar), it has good growth characteristics andproduces relatively high yields of ethanol. For example, in continuousculture at pH 7.0, 65° C., dilution rate of 0.1 h⁻¹, and 50 g/L glucosefeed, the cells produce up to 20 g/L of ethanol (i.e. 0.4 g of ethanol/gof glucose utilised) for 200 hours or more without any drop in ethanolyield.

Strain LN-S (J8) has been deposited with the NCIMB under the terms ofthe Budapest Treaty under accession number NCIMB 41040.

EXAMPLE 4 Production of Strain LN-D

This strain was produced by a double crossover homologous recombinationevent between a linear insertion cassette and the ldh gene from strainLN (see FIG. 4). The insertion cassette (pUC-IC) is a non-replicatingpUC18 plasmid harbouring a kanamycin resistance gene flanked by the ldhand lactase permease (lp) gene sequences. Recombination inactivated boththe ldh and lp genes resulting in a lactate negative phenotype. Thisstrain is also sporulation deficient and resistant to kanamycin. StrainLN-D can tolerate high sugar concentrations (with up to 10 g/L ofresidual sugar), it has good growth characteristics and producesrelatively high yields of ethanol. For example, in continuous culture atpH 7.0, 52° C., dilution rate of 0.1 h⁻¹, and 50 g/L glucose feed, thecells produce up to 20g/L of ethanol (i.e. 0.4 g of ethanol/g of glucoseutilised) for 200 hours or more without any drop in ethanol yield orcell viability. Furthermore, kanamycin selection was not required tomaintain the ldh gene inactivation.

Strain LN-D has been deposited with the NCIMB under the terms of theBudapest Treaty under accession number NCIMB 41041.

EXAMPLE 5 Production of Strain LN-D11

The resistance of strain LN-D to kanamycin was cured after repeatedsubculture to produce strain LN-D11. This strain is lactate negative,sporulation deficient and sensitive to kanamycin concentrations inexcess of 5 μg/ml. Strain LN-D11 can tolerate high sugar concentrations(with up to 10 g/L of residual sugar), it has good growthcharacteristics and produces relatively high yields of ethanol. Forexample, in continuous culture at pH 7.0, 52° C., dilution rate of 0.1h⁻¹, and 50 g/L glucose feed, the cells produce up to 20 g/L of ethanol(i.e. 0.4 g of ethanol/g of glucose utilised) for 200 hours or morewithout any drop in ethanol yield or cell viability. This strain is anideal host for heterologous gene expression.

EXAMPLE 6 Production of Strain LN-DP1

Strain LN-DPI was produced from strain LN-D11 after transformation withthe replicative plasmid pBST22-zym (also referred to as pZP1). Thebackbone of this vector is pBST22 (originally developed by Liao et al(1986) PNAS (USA) 83: 576-580) with the entire pdc gene from Z. mobilisunder the control of the ldh promoter sequence from B.stearothermophilus NCA 1503. Strain LN-DP1 is sporulation deficient;kanamycin resistant, lactate negative and contains the heterologous pdcgene from Z. mobilis. Strain LN-DP1 can utilise high sugarconcentrations, it has good growth characteristics and producesrelatively high yields of ethanol. For example, in continuous culture atpH 7.0, 60° C., dilution rate of 0.1 h⁻¹, and 50g/L glucose feed, thecells produce up to 25g/L ethanol (i.e. 0.5 g of ethanol/g of glucoseutilised) for 200 hours or more without any drop in ethanol yield.

EXAMPLE 7 Production of Strain TN

Strain TN arose spontaneously from T13 during nitrogen adaptation incontinuous culture. This strain is more robust than the parental strainunder vigorous industrial conditions (i.e. low pH, high sugarconcentrations, and in crude hydrolysate feed stocks) and is moreamenable to plasmid transformation, making it an ideal host for geneticengineering. Strain TN is sporulation deficient and lactate negative(the ldh gene has been inactivated by transposon mutagenesis into thecoding region of the ldh gene). Strain TN is a good ethanol producer ondilute sugar feeds. For example, in continuous culture at pH 7.0, 70°C., dilution rate of 0.1 h⁻¹, and 20 g/L glucose, the cells produce upto 8 g/L ethanol (i.e. 0.4 g of ethanol/g of glucose utilised) for 100hours or more without any drop in ethanol yield. This strain issensitive to kanamycin concentrations in excess of 5 μg/ml and an idealhost for genetic manipulation.

Strain TN has been deposited at the NCIMB under the terms of theBudapest Treaty under accession number NCIMB 41039.

EXAMPLE 8 Production of Strain TN-P1

Strain TN-P1 was produced from strain TN after transformation with thereplicative plasmid pBST22-zym. As previously described, pBST22-zym wasproduced by the incorporation of a pdc gene in vector pBST22. Thetransformation efficiency of TN was 10-fold higher with pBST22-zym thanpBST22 and the colony size of the transformants were significantlylarger indicating that pdc expression conferred a growth advantage tothe cells.

TN-P1 is also more stable and a better ethanol producer than TN,especially in sugar concentrations greater than 20 g/L. For example, incontinuous culture controlled at pH 7.0, 52° C. with a dilution rate of0.1 h⁻¹, and a sugar feed containing 20-50 g/L, strain TN-P1 produced upto 0.5 g ethanol/g sugar utilised for 600 hours with no significant dropin yield or cell viability. In addition, in continuous culturecontrolled at pH 7.0, 52° C. with a dilution rate of 0.1 h⁻¹, and awheat crude hydrolysate feed, TN-P1 produced 0.4 g ethanol/g sugarutilised in for 400 hours with no significant drop in ethanol yield orculture viability.

Plasmid selection in strain TN-P1 was first maintained with kanamycinbut the plasmid was found to be relatively stable without selection andno significant plasmid loss was detected after 300 hours of continuousculture. The plasmid and PDC enzyme were found to be relatively stableat high temperatures. In continuous culture controlled at pH 7.0, with adilution rate of 0.1 h⁻¹, and a glucose feed of 30 g/L, there was nosignificant drop in ethanol yield or culture viability when the growthtemperature was increased from 52 to 60° C.

EXAMPLE 9 Production of Strain TN-P3

Strain TN-P3 was produced from strain TN after transformation with thereplicative plasmid pFC1-PDC1. The backbone of this vector is pFC1 (FIG.8) which was formed from a fusion of pAB124 (Bingham et al., Gen.Microbiol., 114, 401-408, 1979) and pUC18. The pdc gene was amplifiedfrom Z. mobilis chromosomal DNA by PCR using the following primers. Therestriction sites BamHI and SacI (underlined) were introduced into theamplified gene. 5′-GAGCTCGCAATGAGTTATACTGTC-3′5′-GGATCCCTAGAGGAGCTTGTTA-3′

The ldh promoter was amplified by PCR from Bacillus LN using thefollowing primers. The restriction sites SacI and BamHI (underlined)were introduced into the amplified sequence.5′-GGATCCGGCAATCTGAAAGGAAG-3′ 5′-GAGCTCTCATCCTTTCCAAAA-3′

The ldh promoter sequence and pdc gene were digested with SacI and BamHIand then ligated together (FIG. 6). The construct was then digested withBamHI and ligated into BamHI digested pFC1 to form plasmid pFC1-PDC1(FIG. 9).

Strain TN-P3 is a good ethanol producer and produces yields in excess of0.45 g ethanol/g sugar at temperatures between 50 and 60° C.

Gene Inactivation

Single-Crossover Recombination (SCO) (FIG. 31

SCO or Campbell-type integration was used for directed ldh geneinactivation. An internal fragment (700 bp) of the target gene (ldh) wasfirst cloned into pUBUC to form pUBUC-LDH.

Plasmid pUBUC is a shuttle vector for DNA transfer between Escherichiacoli and Bacillus strains LN and TN and was formed from the fusion ofpUB110 and pUC18. This vector contains a selectable marker that confersresistance to kanamycin and a Gram-positive and Gram-negative replicon.The plasmid is temperature sensitive in Bacillus and cannot replicateabove 54° C.

Plasmid pUBUC-LDH was first methylated (in vivo) and then transformedinto the host strain (LN) at the permissible temperature (50° C.) forplasmid propagation. The growth temperature was then increased to 65°C., preventing plasmid replication and integrants were selected usingkanamycin. Integration of the plasmid DNA into the ldh gene resulted ingene inactivation and a lactate negative phenotype.

Double-Crossover Recombination (DCO) (FIGS. 4 & 5)

DCO or replacement recombination differs from SCO in that it results inintegration of only one copy of the target DNA and typically, a regionof chromosomal DNA is replaced by another region, either foreign DNA ormutationally altered homologous DNA. The target DNA (kanamycin marker)was flanked on either side by mutationally altered fragments of the ldhgene in plasmid pUC-IC (a non-replicative vector based on pUC18). Thevector was first methylated (in vivo) and then linearised at a siteoutside the flanking region (this prevents SCO). The methylated,linearised plasmid DNA was then transformed into strain LN andintegrants were selected using kanamycin.

This technique has also be used to inactivate ldh and integrate a copyof the pdc gene into the chromosome simultaneously (FIG. 5) and can beapplied to other genes of interest.

EXAMPLE 10 PDC Expression

In the expression plasmid pBST22-zym, the pdc gene from Z. mobilis isunder the control of the ldh promoter sequence from B.stearothermophilus NCA1503 (FIG. 6). This plasmid was transformed intostrain TN to form TN-P1. Although PDC improved cell growth, pdcexpression and subsequent enzyme activity was relatively wreak and therewas only a small increase in ethanol yield. In addition, PDC activity issensitive to temperature and rapidly declined at growth temperaturesgreater than 60° C.

Therefore, we increased expression of pdc by firstly replacing the ldhpromoter from B. stearothermophilus NCA 1503 with the ldh promotersequence from Bacillus sp. LN (FIG. 6). The pdc gene under the controlof ldh promoter from strain LN (construct 2) was subcloned into pFC1 toform plasmid pFC1-PDC1 (FIG. 9). Plasmid pFC1 is a shuttle vector thatcontains a Gram-positive and Gram-negative replicon and confersresistance to ampicillin and tetracycline. This plasmid was transformedinto strain TN to form TN-P3.

Strain Performance

TN-P3 produces significantly more ethanol than the untransformed controlstrain TN. Strain Ethanol Concentration TN 21.5 mM TN-P1 24.0 mM TN-P339.5 mM

The strains were cultured in a culture medium containing JSDsupplemented with 50 mM PIPES buffer and 2% glucose at 54° C. for 24hours. TN-P1 and TN-P3 cultures were supplemented with kanamycin (12μg/ml) and tetracycline (10 μg/ml), respectively.

The thermostability of pdc can be improved by cloning an alternative pdcgene, encoding a more thermostable PDC enzyme, from Saccharomycescerevisiae. This gene, referred to as pdc5 will also be under thecontrol of the ldh promoter from strain LN (see FIG. 9, construct 3).The construct will be cloned into pFC1 and the resulting plasmidpFC1-PDC5 will be transformed into strain TN.

Development of an Artificial PDC Operon

An artificial PDC operon can be constructed using interchangeable genesequences from the ldh promoter, pdc genes from Z. mobilis and the yeastSaccharomyces cerevisiae, and adh from LN (see FIG. 10).

-   1) pdc expression should be increased when the ldh promoter from B.    stearothermophilus NCA1503 is replaced by the ldh promoter from LN    and will result in higher ethanol yields.-   2) the ethanol yields should be increased further if adh from LN is    co-expressed with pdc from Z. mobilis in a PDC operon Ethanol yields    should be close to theoretical values of 0.5 g ethanol/g sugar.-   3) the thermostability of pdc may be increased above 64° C. if    the Z. mobilis pdc gene is replaced by the pdc5 gene from S.    cerevisiae. This will increase the growth temperature from 64 to 70°    C.    Integration of the PDC Operon into Strain LN-D11

By fusing the PDC operon to the insertion element sequence (firstidentified in the ldh gene from TN) several copies of the PDC operon canbe introduced into multiple sites on the chromosome increasing both thestability and gene dosage.

This expression strategy may also be used for other genes of interest.

1. A Gram-positive bacterium which has been transformed with aheterologous gene encoding pyruvate decarboxylase (pdc), but has solelynative alcohol dehydrogenase (adh) function, and does not comprise agene encoding a functional lactate dehydrogenase, wherein the bacteriumis a thermophile and is a Bacillus sp.
 2. The Gram-positive bacteriumaccording to claim 1 wherein the Bacillus is selected from the groupconsisting of: B. stearothermophilus, B. caldovelox, B. caldotenax, B.thermoglucosidasius, B. coagulans, B. licheniformis, B.thermodenitrificans, and B. caldolyticus.
 3. The Gram-positive bacteriumaccording to claim 1 or 2 wherein the bacterium comprises a geneencoding lactate dehydrogenase and the gene has been inactivated.
 4. TheGram-positive bacterium according to claim 3 in which the lactatedehydrogenase gene has been inactivated by homologous recombination. 5.The Gram-positive bacterium according to claim 1 in which theheterologous gene is from Zymomonas sp or from Saccharomyces cerevisiae.6. The Gram-positive bacterium according to claim 5 in which theheterologous gene is from Z. mobilis.
 7. The Gram-positive bacteriaaccording to claim 5 in which the heterologous gene is pdc 5 from S.cerevisiae.
 8. The Gram-positive bacterium according to claim 7 whereinthe heterologous gene is incorporated into the chromosome of thebacterium.
 9. The Gram-positive bacterium according to claim 1 in whichthe bacterium has been transformed with a plasmid comprising theheterologous gene.
 10. The Gram-positive bacterium according to claim 9,wherein the plasmid is pFC1.
 11. The Gram-positive bacterium accordingto claim 1, wherein the heterologous gene is operatively linked to thelactate dehydrogenase promoter from Bacillus strain LN (NCIMB accessionnumber 41038).
 12. A bacterial strain selected from the group consistingof: Strains LN (NCIMB accession number 41038); TN (NCIMB accessionnumber 41039); LN-S (J8) (NCIMB accession number 41040); and LN-D (NCIMBaccession number 41041).
 13. A bacterium of strain LN, (NCIMB accessionnumber 41038) that has undergone a spontaneous transposon mutation toinsert a transposon into the coding region of the lactate dehydrogenasegene, the bacterium being non-sporulating.
 14. A bacterium of strain TN(NCIMB accession number 41039) that has been transformed with plasmidpBST22-zym.
 15. A bacterium of strain TN (NCIMB accession number 41039)that has been transformed with plasmid pFC1-PDC1.
 16. A bacterium ofstrain LN-D (NCIMB accession number 41041) that has undergone aspontaneous mutation and is lactate negative and sporulation deficient.17. The bacterium according to claim 16 that has been transformed withplasmid pBST22-zym.
 18. A method of producing ethanol comprisingculturing a bacterium according to claim 1 or a strain according to anyone of claims 12 to 17 under suitable conditions.
 19. The methodaccording to claim 18 in which the culturing is at a temperature between40-75° C.
 20. The method according to claim 19 in which the culturing isat a temperature of 52-65° C.
 21. The method according to claim 19 inwhich the culturing is at a temperature of 60-65° C.
 22. Plasmid pFC1.23. Plasmid pFC1-PDC1.